The peptidoglycan (PG) cell wall has long been an attractive target for antibiotic intervention since the late stages of its synthesis take place on he solvent accessible surface of bacterial cells. This essential macromolecule defines bacterial size and shape and provides cells with mechanical strength to resist cell envelope breakdown. Additionally, recent research has demonstrated the importance of the spatiotemporal coordination of PG biosynthesis for bacterial growth, revealing a vulnerability that can be exploited for the development of new antibiotics. The development of methods to enable spatiotemporal tracking of PG synthesis in live bacterial cells is critical to advancing the understanding of the mechanisms of PG synthesis dynamics. In the absence of such methods, identification of new antibiotic targets or identification of novel antibiotic agents will remain elusive. This project has three specific aims that are focused on a long-term goal of elucidating the mechanisms of PG synthesis dynamics. The first Specific Aim seeks to design and develop a series of D-amino acid- and dipeptide-based fluorogenic probes, with optimized photophysical properties, that when coupled with integrated nanochannel and microfluidic devices, and automated image analysis tools, will propel the study of PG dynamics to an unprecedented level of spatiotemporal resolution. The subsequent specific aims will utilize these tools and approaches to analyze the mechanisms of PG dynamics in bacterial model systems with differing cell shapes and cell envelope architectures. In Specific Aim 2, the probes and methods developed under specific aim 1 will be employed to test two major and long-standing hypotheses regarding the spatiotemporal coordination of the elongation and division PG synthesis machineries as well as the coordination between PG hydrolysis and synthesis in the principal model for ovoid-shaped cells, Streptococcus pneumoniae. In Specific Aim 3, PG spatiotemporal dynamics will be examined at an unprecedented resolution for the major model species for rod-shaped Gram negative bacteria with a thin layer of PG, E. coli, and for rod-shaped Gram-positive bacteria with a thick layer of PG in the model organism, B. subtilis. Furthermore, Aim 3 will leverage a high-throughput microscopy screening platform, the availability of a comprehensive strain collection in which each gene has been separately deleted, and the powerful genetics of both species, to systematically and randomly screen for genes involved in PG synthesis dynamics. The comparative analysis of the three model systems will identify the core principles of PG dynamics and how they can be modified to yield different outcomes in dynamics, cell shape and cell envelope architecture. Aims 2 and 3 will feed back into Aim 1 and lead to the design of improved probes and nanochannel configurations. The highly integrated approach, coupled with the individual expertise of the investigators, will provide an unprecedented understanding of PG synthesis and dynamics that can be used to uncover new antibacterial targets, an important step toward addressing the critical need for the discovery of new antibiotics.